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Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

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Mutations of F401 and cooperative gating: Cole-Moore analysis. (A) Families of currents from the wt, F401L, and F401A channels were obtained as follows: pulses to 0 mV were preceded by 1-s-long prepulses to voltages between −140 and −40 mV (wt, left), −170 and −50 mV (F401L, center), or between −130 and −20 mV (F401A, right). A few of the most depolarizing prepulses are in the activation range of the channels, as evident from the steady current recorded during the prepulse. The most negative prepulses induce greatest delay at the onset of activation. (B) The voltage families above were scaled in amplitude to match at the peak to compensate for steady state C-type inactivation incurred at the more depolarized prepulses. Scaled traces were then shifted along the time axis to obtain the best superposition. Note that, whereas in wt and F401L (left, center) traces in which the prepulse opens channels with detectable probability are not superimposable on the rest of the family, in F401A such behavior is not seen. At the onset of current during the main pulse, gating currents can be seen as bumps in the F401A family.
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Figure 11: Mutations of F401 and cooperative gating: Cole-Moore analysis. (A) Families of currents from the wt, F401L, and F401A channels were obtained as follows: pulses to 0 mV were preceded by 1-s-long prepulses to voltages between −140 and −40 mV (wt, left), −170 and −50 mV (F401L, center), or between −130 and −20 mV (F401A, right). A few of the most depolarizing prepulses are in the activation range of the channels, as evident from the steady current recorded during the prepulse. The most negative prepulses induce greatest delay at the onset of activation. (B) The voltage families above were scaled in amplitude to match at the peak to compensate for steady state C-type inactivation incurred at the more depolarized prepulses. Scaled traces were then shifted along the time axis to obtain the best superposition. Note that, whereas in wt and F401L (left, center) traces in which the prepulse opens channels with detectable probability are not superimposable on the rest of the family, in F401A such behavior is not seen. At the onset of current during the main pulse, gating currents can be seen as bumps in the F401A family.

Mentions: We sought to confirm further that the mutations at F401 alter the deviation from independence seen in wt channels upon entering the open state. As originally described by Cole and Moore 1960, multistep activation gating results in greater delay in current turn-on when the channels are subjected to progressively more negative voltages before the test pulse. The greater delay occurs as the equilibrium distribution of channels among closed states shifts in favor of the states most distant from the open state. For an independent gating scheme, a family of current waveforms corresponding to different prepulse voltages becomes superimposable simply by a translation along the time axis that allows for the amount of delay lost or gained. These transformations of wt, F401L, and F401A currents are shown in Fig. 11. In wt, over the voltage range between −140 and −70 mV, there is almost no current activation during the prepulse, and the corresponding traces at 0 mV are parallel and superimposable by a shift along the time axis. However, at the prepulse voltages of −50 and −40 mV, wt channels open with nonnegligible probability and the 0-mV current waveforms cannot be superimposed on the rest of the family by this procedure (Fig. 11 B, left; Zagotta et al. 1994a). This property is also observed with F401L channels (Fig. 11, middle). In contrast, in F401A, the Cole-Moore shift is present with complete superimposability over the prepulse voltage range of −130 to −20 mV (Fig. 11, right). During the more depolarized prepulses in this family, F401A channels are significantly activated, but their entry into the open state confers no new kinetic features to the 0-mV traces that would suggest nonindependent subunit behavior.


Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Mutations of F401 and cooperative gating: Cole-Moore analysis. (A) Families of currents from the wt, F401L, and F401A channels were obtained as follows: pulses to 0 mV were preceded by 1-s-long prepulses to voltages between −140 and −40 mV (wt, left), −170 and −50 mV (F401L, center), or between −130 and −20 mV (F401A, right). A few of the most depolarizing prepulses are in the activation range of the channels, as evident from the steady current recorded during the prepulse. The most negative prepulses induce greatest delay at the onset of activation. (B) The voltage families above were scaled in amplitude to match at the peak to compensate for steady state C-type inactivation incurred at the more depolarized prepulses. Scaled traces were then shifted along the time axis to obtain the best superposition. Note that, whereas in wt and F401L (left, center) traces in which the prepulse opens channels with detectable probability are not superimposable on the rest of the family, in F401A such behavior is not seen. At the onset of current during the main pulse, gating currents can be seen as bumps in the F401A family.
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Related In: Results  -  Collection

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Figure 11: Mutations of F401 and cooperative gating: Cole-Moore analysis. (A) Families of currents from the wt, F401L, and F401A channels were obtained as follows: pulses to 0 mV were preceded by 1-s-long prepulses to voltages between −140 and −40 mV (wt, left), −170 and −50 mV (F401L, center), or between −130 and −20 mV (F401A, right). A few of the most depolarizing prepulses are in the activation range of the channels, as evident from the steady current recorded during the prepulse. The most negative prepulses induce greatest delay at the onset of activation. (B) The voltage families above were scaled in amplitude to match at the peak to compensate for steady state C-type inactivation incurred at the more depolarized prepulses. Scaled traces were then shifted along the time axis to obtain the best superposition. Note that, whereas in wt and F401L (left, center) traces in which the prepulse opens channels with detectable probability are not superimposable on the rest of the family, in F401A such behavior is not seen. At the onset of current during the main pulse, gating currents can be seen as bumps in the F401A family.
Mentions: We sought to confirm further that the mutations at F401 alter the deviation from independence seen in wt channels upon entering the open state. As originally described by Cole and Moore 1960, multistep activation gating results in greater delay in current turn-on when the channels are subjected to progressively more negative voltages before the test pulse. The greater delay occurs as the equilibrium distribution of channels among closed states shifts in favor of the states most distant from the open state. For an independent gating scheme, a family of current waveforms corresponding to different prepulse voltages becomes superimposable simply by a translation along the time axis that allows for the amount of delay lost or gained. These transformations of wt, F401L, and F401A currents are shown in Fig. 11. In wt, over the voltage range between −140 and −70 mV, there is almost no current activation during the prepulse, and the corresponding traces at 0 mV are parallel and superimposable by a shift along the time axis. However, at the prepulse voltages of −50 and −40 mV, wt channels open with nonnegligible probability and the 0-mV current waveforms cannot be superimposed on the rest of the family by this procedure (Fig. 11 B, left; Zagotta et al. 1994a). This property is also observed with F401L channels (Fig. 11, middle). In contrast, in F401A, the Cole-Moore shift is present with complete superimposability over the prepulse voltage range of −130 to −20 mV (Fig. 11, right). During the more depolarized prepulses in this family, F401A channels are significantly activated, but their entry into the open state confers no new kinetic features to the 0-mV traces that would suggest nonindependent subunit behavior.

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH
Related in: MedlinePlus